Inhibition by α-tocopherol and L-ascorbate of linoleate hydroperoxidation and β-carotene bleaching activities in durum wheat semolina

In order to gain some insights into durum wheat lipoxygenase (LOX) biochemistry and in particular in order to find out whether the antioxidants α-tocopherol and L-ascorbate can prevent the LOX-dependent semolina bleaching, which leads to a low quality of pasta product, a study of LOX activity in a crude protein extract from semolina was performed by means of spectrophotometric techniques. Measurements made of linoleate hydroperoxidation reaction (λ = 234 nm) and of the coupled β-carotene bleaching reaction (λ = 460 nm), demonstrated the existence of more than one enzyme exhibiting LOX activity as revealed by the different pH profiles. The linoleate hydroperoxidation reaction exhibited a sigmoidal dependence on the substrate concentration (K0.5 = 60 ± 0.67 μM, V(max) = 1.12 ± 0.055 units/g of semolina), while β-carotene bleaching showed a hyperbolic relationship and followed the Michaelis-Menten equation (K(m) = 3.3 ± 0.53 μM, V(max) = 0.030 ± 0.0062 units/g). Both linoleate hydroperoxidation and β-carotene bleaching were strongly inhibited by added α-tocopherol. In both cases, the enzyme affinity for the substrate was reduced, while the V(max) was not affected. In particular, β-carotene bleaching was inhibited in a competitive manner (K(i) = 7.7 ± 0.21 μM). L-ascorbate also inhibited both reactions: The V(max) of linoleate hydroperoxidation was lowered, while the K0.5 remained unchanged; in the case of β-carotene bleaching the inhibition was uncompetitive (K(i) = 445 ± 71.5 μM). Therefore, the use of both α-tocopherol and L-ascorbate to reduce semolina bleaching during pasta making is suggested. A purification procedure of semolina LOX is also reported, showing the existence of two isoforms. (C) 2000 Academic Press.